TO BE HANDED IN LAB 2 - 10 points
The first part of the assignment is to create a figure and to write a brief analysis of the active site of the β-galactosidase molecule. The figure will be made from an image that you will generate during your RasMol modeling exercise described in RasMol Tutorial. This image should include two bound ONPG substrate molecules (their code number is 2001, 2002 in RasMol), magnesium ions, a sodium ion, and the surrounding amino acids that form the geometry of the binding site. Make sure the catalytically significant amino acids in the active site are labeled or highlighted in some way. In order to find out which of the amino acids are the most important, please read the following article: Jacobson RH, Zhang X-J, DuBose RF, Matthews BW (1994), Three-dimensional structure of β-galactosidase from E. Coli. Nature 369; 761-766. It can be found from the Wellesley Science library’s electronic journals using the link: http://0-www.nature.com.luna.wellesley.edu/nature/journal/v369/n6483/pdf/369761a0.pdf
It will also be helpful to look at the figures generated by Jacobsen and other researchers who have modeled the active site of this enzyme (Jacobsen et al. 1994, Juers et al. 2001). Make sure the background color is white on your final submitted image. If you save your image as a GIF file (under Export in the RasMol menu) you can import it into a Microsoft word document. Write a properly formatted legend for your figure.
In addition to the figure image, you will submit a 1-3-page discussion of the molecular modeling and sequence analysis work done today. This discussion should be e a clear, accurate, and literature supported analysis of both the general structure of the active site and of the specific amino acids that are crucial to its formation or function. You should also include the chemical character of the crucial elements and identify the amino acids that co-ordinate with essential ions. In addition, there should be an explanation of where and why there are highly conserved amino acids. The consensus information will come from the sequence analysis performed using ClustalW described in the ClustalW Tutorial. Be sure to end your discussion with a list of references used (and cited) in your discussion and those used in making the figure (cited in the legend). Please use CSE (Council of Science Educators) name/year citation style for formatting your source citations in this and ALL the written work for this course. For more information on proper CSE citation style, see: http://www.wsulibs.wsu.edu/electric/quickguides/docs/CSEhome.html.
Jacobson RH, Zhang X-J, DuBose RF, Matthews BW (1994) Three-dimensional Structure of β-galactosidase from E. Coli. Nature 369; 761-766.
Juers DH, Heightman TD, Vasella A, McCarter JD, Mackenzie L, Withers SG, Matthews BW (2001) A structural view of the action of Escherichia coli (lacZ) β-galactosidase. Biochemistry. 40: 14781-94.
Roth NJ, Rob B, Huber RE (1998) His–357 of β-galactosidase (Escherichia coli) Interacts with the C3 Hydroxyl in the Transition State and Helps To Mediate Catalysis. Biochemistry 37: 10099-10107.
|| At or Above Standard
|| Below Standard
|| Points Earned
|| 2-3 pts. Figure is well designed to show main points made in the discussion. Figure includes 2 substrate molecules & other non-protein molecules that are important. Figure labels or color codes the amino acids mentioned in the discussion and is oriented to distinguish as many as possible.
|| 0-1.5 Figure is not well designed or oriented to illustrate points mentioned in discussion. Figure is missing essential non-protein molecules or substrate molecules. Figure is missing labels or some form of color coding of as many as possible of the amino acids mentioned in discussion.
| Figure Legend
|| 0.5-1 Figure legend is below figure & includes a number. Legend includes all essential information and no unnecessary detail about how figure was generated and what the figure illustrates. Legend does not include results or other material appropriate for the discussion.
|| 0-0.5 Missing legend. Legend is in wrong place or does not include a fig. number. Missing information about how figure was generated (RasMol). Missing part or all of key to symbols/ colors. Missing information that adequately identifies what the figure illustrates. Legend includes unimportant detail &/or explanatory material appropriate for the discussion but not for the legend.
|| 4-5 Includes a clear, accurate, and literature supported discussion of both the general structure of the active site and of specific amino acids crucial to its formation or function. Includes the chemical character of the crucial elements & which amino acids co-ordinate with essential ions. Includes explanation of most conserved amino acids from ClustalW
|| 0-3.5 Missing, incorrect, or unsupported information about specific amino acids or non-protein molecules crucial to active site. Missing or incorrect information about the chemical character of the amino acids and non-protein molecules discussed. Missing information from ClustalW about the most conserved amino acids or parts of the protein.
|| 0.5-1 Uses an adequate number of appropriate sources and cites those sources, including RasMol and ClustalW, properly in the body of the paper as well as in the reference list.
|| 0-0.5 Does not use an adequate number or appropriate sources. Does not cite material from sources in proper format in the body of the paper. Does not include a properly formatted reference list at the end of the discussion.